During fasting, elevations in circulating pancreatic glucagon promote hepatic glucose output through induction of the gluconeogenic program by the CREB coactivator TORC2. Sequestered in the cytoplasm during feeding, TORC2 translocates to the nucleus in response to fasting signals, where it triggers gluconeogenic gene expression in concert with the Forkhead transcription factor FOXO1. Preliminary studies indicate that TORC2 is transiently activated through P300-dependent acetylation during early fasting, when it stimulates the gluconeogenic program via an association with a histone methyl-transferase complex. TORC2 is silenced through SIRT1-mediated deacetylation during prolonged fasting, when FOXO1 is reciprocally activated. Three Aims are proposed: In Aim I, the role of P300 in augmenting TORC2 activity through acetylation during early fasting will be determined. Acetylation sites in TORC2 will be identified, and the importance of P300 in catalyzing TORC2 acetylation will be evaluated. The role of the NAD+ dependent deacetylase SIRT1 in silencing TORC2 through deacetylation during prolonged fasting will also be explored. In Aim II, the importance of the Ser/Thr kinase SIK2 in modulating hepatic TORC2 activity through phosphorylation of P300 will be evaluated. SIK2 phosphorylation sites in P300 will be identified, and their role in disrupting the P300:TORC2 interaction and thereby reducing TORC2 acetylation will be analyzed. In Aim III, the role of a TORC2 associated histone methyl-transferase (HMT) complex in mediating induction of the gluconeogenic program during fasting will be determined. The importance of TORC2 for recruitment of HMT complexes and for histone methylation over gluconeogenic promoters during fasting will be determined, by depletion of TORC2 or HMT components, and by expression of HMT interaction-defective mutant TORC2 proteins. The potential role of HMTs in modulating gluconeogenic gene expression by methylating TORC2 will also be evaluated through identification and mutation of relevant sites in TORC2. Taken together, the proposed studies will provide insight into regulatory pathways that modulate fasting metabolism through a coactivator that is required for glucose balance and that contributes to hyperglycemia in diabetes. The results may lead to the identification of new molecular targets for the development of therapeutic compounds that improve glucose control in insulin resistant individuals. PUBLIC HEALTH RELEVANCE: The pancreatic hormone glucagon maintains circulating glucose levels during fasting by turning on a genetic switch, called TORC2, that increases glucose production in the liver; insulin protects against abnormal elevations in blood glucose during feeding by turning off the TORC2 switch. Glucagon and insulin exert these opposing effects on the TORC2 switch through a group of enzymes that cause different chemical changes in the TORC2 protein. By characterizing these chemical changes in TORC2 and understanding how they modify the ability for this switch to trigger glucose production, our studies may lead to new therapies for the treatment of diabetic patients.